Intramolecular cross-linking of the extrinsic 33-kDa protein leads to loss of oxygen evolution but not its ability of binding to photosystem II and stabilization of the manganese cluster.
The extrinsic 33-kDa protein of photosystem II (PSII) was intramolecularly cross-linked by a zero-length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The resulting cross-linked 33-kDa protein rebound to urea/NaCl-washed PSII membranes, which stabilized the binding of manganese as effectively as the untreated 33-kDa protein. In contrast, the oxygen evolution was not restored by binding of the cross-linked protein, indicating that the binding and manganese-stabilizing capabilities of the 33-kDa protein are retained but its reactivating ability is lost by intramolecular cross-linking of the protein. From measurements of CD spectra at high temperatures, the secondary structure of the intramolecularly cross-linked 33-kDa protein was found to be stabilized against heat treatment at temperatures 20 degrees C higher than that of the untreated 33-kDa protein, suggesting that structural flexibility of the 33-kDa protein was much decreased by the intramolecular cross-linking. The rigid structure is possibly responsible for the loss of the reactivating ability of the 33-kDa protein, which implies that binding of the 33-kDa protein to PSII is accompanied by a conformational change essential for the reactivation of oxygen evolution. Peptide mapping, N-terminal sequencing, and mass spectroscopic analysis of protease-digested products of the intramolecularly cross-linked 33-kDa protein revealed that cross-linkings occurred between the amino group of Lys48 and the carboxyl group of Glu246, and between the carboxyl group of Glu10 and the amino group of Lys14. These cross-linked amino acid residues are thus closely associated with each other through electrostatic interactions.
Enami
I
I
Department of Biology, Faculty of Science, Science University of Tokyo, Kagurazaka 1-3, Shinjuku-ku, Tokyo 162, Japan. enami@rs.noda.sut.ac.jp
Kamo
M
M
Ohta
H
H
Takahashi
S
S
Miura
T
T
Kusayanagi
M
M
Tanabe
S
S
Kamei
A
A
Motoki
A
A
Hirano
M
M
Tomo
T
T
Satoh
K
K
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D008345:Manganese / Q000378:metabolism*; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases / Q000378:metabolism; D018724:Spinacia oleracea
D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D008345:Manganese; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D010100:Oxygen